The mating type loci in Saccharomyces cerevisiae provide a striking example of position effect regulation. Mating type regulatory genes located at the ends of chromosome III in yeast in loci designated HML and HMR are packaged in heterochromatin, which precludes their expression. We plan to explore the mechanism by which heterochromatin is deposited and maintained at silent loci and to examine the structure of the heterochromatin to determine how it precludes access to the transcriptional apparatus. Our experimental plan includes (1) determination of the relationship between DNA replication and heterochromatin formation; (2) an in vivo analysis of chromatin assembly factors; (3) isolation and analysis of histone mutants that enhance heterochromatin persistence; and (4) isolation and characterization of silenced minicircles. These results should shed light on regulatory processes affecting such diverse events as X-inactivation and chromosome imprinting, which underlies various diseases including a number of sporadic cancers. We also plan to examine the mechanism underlying directionality of mating type interconversion in yeast. Activation of information within two silent mating type cassettes occurs by directed transposition to an expression locus. Choice of donor cassettes is not random but is dictated by the mating type of the cell through activation or inhibition of a site on the chromosome that activates the entire distal portion of the arm for recombination. We plan to explore the mechanistic basis of this selective interaction of distant regions of the genome by (1) determining the large scale organization of chromosome III in vivo; and (2) determining the proteins required for activation of the chromosome for recombination. The results of these experiments should help us define how distant regions of a genome can functionally and selectively interact in execution of a developmental program. These observations should yield insights in similar processes, such as immunoglobulin class switching, in larger eucaryote.